Engineering

Isoorientin is a natural flavonoid compound that is widely present in many foods and possesses various functional activities, including reducing the risk of chronic diseases caused by oxidation, inflammation, and cancer cell proliferation. Its remarkable hypoglycemic effect is particularly significant, offering potential as a new treatment for metabolic diseases such as type II diabetes. Our goal is to mass-produce Gentian triflorum C-glycosyltransferase (Gt6CGT), which can convert luteolin into isoorientin. This approach aims to provide a low-cost synthesis pathway for isoorientin.

Design

We use the Escherichia coli prokaryotic expression system to obtain the recombinant Gt6CGT protein. The constructed Gt6CGT_pET-21a(+) plasmid is transformed into $E. coli$ Rosetta cells. SDS-PAGE analysis, Gt6CGT protein activity assays, and HPLC detection are then conducted to analyze the production of isoorientin. (Figure 1).

Build

Through literature research, we identified a glycosyltransferase (Gt6CGT) in Gentian triflora. We used GenScript to synthesize the relevant sequence and cloned it into the pET-21a(+) vector. Using seamless cloning technology (Figure 2), we constructed the recombinant plasmid Gt6CGT_pET-21a(+) (Figure 3).

Figure 2. Ligation reaction of Gt6CGT_pET-21a (+) by seamless cloning.

Figure 3. Recombinant Plasmid Map of Gt6CGT_pET-21a(+)

First, the pET21-a(+) plasmid was digested with BamHI, and the Gt6CGT fragment containing homologous arms of the pET-21a(+) vector was amplified by PCR. The digestion products and PCR products were analyzed using 1.0% agarose gel electrophoresis (Figure 4), and the DNA was recovered. Then, the target gene fragment and the linearized vector were recombined using a seamless cloning kit. The recombinant product was transformed into $E. coli$ TOP10 competent cells. Colony PCR (Figure 5) and sequencing results (Figure 6) confirmed that the Gt6CGT gene was successfully inserted into the pET-21a(+) vector. In Figure 6, the middle sequence indicated by the two arrows is the full length of the Gt6CGT sequence.The correctly sequenced plasmid was then transferred into $E. coli$ Rosetta cells.

Figure 4. PCR amplification of Gt6CGT (high fidelity enzyme) and digestion of pET-21a vector

Figure 5. Colony PCR identification of TOP10 cells

Figure 6. Sequencing results of recombinant plasmid

Test

1. Protein expression

We used SDS-PAGE to analyze the expression of Gt6CGT after induction with 0.1 mM IPTG. As shown in Figure 7, compared to the uninduced samples (lanes 1 and 4), the protein bands corresponding to IPTG-induced Gt6CGT (lanes 2-3 and 5-7) appear between 45 kDa and 66.2 kDa. The theoretical size of the Gt6CGT protein is 53.4 kDa, indicating that the protein was successfully expressed in Rosetta cells.

Figure 7. SDS-PAGE results of Gt6CGT_pET-21a(+)

1,4: 0mM IPTG；2,3,5,6,7: 0.1 mM IPTG.

2. Activity test of Gt6CGT

After successfully expressing the Gt6CGT protein, we used a microplate reader to assess its enzymatic activity. UDP-glucose was used as the glycosyl donor, and α-naphthol as the glycosyl acceptor. Under the catalytic action of Gt6CGT protein, α-naphthol glycoside was produced. The change in fluorescence intensity at an excitation wavelength of 287 nm and an emission wavelength of 335 nm was measured to reflect the enzyme activity. The experimental results are shown in Figure 8. The CK group used Tris-HCl as a blank control, while the other two groups used the supernatant and inclusion bodies from the sonicated $E. coli$ as enzyme solutions. Compared to the CK group, the fluorescence values of both the supernatant and inclusion body groups significantly increased, with the supernatant group showing higher fluorescence than the inclusion body group. This indicates that the extracted Gt6CGT protein possesses glycosyltransferase activity.

1: PB buffer; 2: supernatant protein; 3: inclusion body protein

3. HPLC detection

The samples were analyzed by high-performance liquid chromatography (HPLC) to detect the presence of isoorientin (Figure 9). The reaction mixture consisted of 1 mM UDP-glucose as the glycosyl donor, 1 mM luteolin as the glycosyl acceptor, 50 mM phosphate buffer (pH 7.5), 1% DMSO (v/v), and an appropriate amount of Gt6CGT, with a total volume of 1000 μL. The reaction was incubated at 50°C for 30 minutes, after which 400 μL of methanol was added to stop the reaction.As shown in Figure 9, isoorientin was detected in both Groups C and D, confirming that Gt6CGT expressed through the $E. coli$ prokaryotic expression system was catalytically active.

Figure 9. Determination of isoorientin by HPLC

A: Isoorientin standard; B: CK-PBS replaces enzyme solution; C: the supernatant of induced bacterial solution after crushing; D: the induced bacterial solution.

Learn

We first amplified the Gt6CGT gene and then constructed the recombinant plasmid using seamless cloning technology. The constructed vector was transformed into $E. coli$ TOP10 cells for amplification. The amplified vector was subsequently transformed into $E. coli$ Rosetta cells. After successfully obtaining transformed Rosetta strains, IPTG was added to induce Gt6CGT protein expression. The SDS-PAGE results confirmed the successful induction of Gt6CGT protein, and fluorescence detection indicated that the Gt6CGT protein possessed glycosyltransferase activity. HPLC analysis further confirmed the formation of isoorientin. Therefore, the Gt6CGT recombinant protein can be used for large-scale production of isoorientin in the future, thereby reducing its production costs.